This project was initiated to analyze, both qualitatively and quantitatively, changes in total cellular protein patterns during cellular differentiation and transformation using the technique of quantitative two-dimensional electrophoresis. A cell culture system has been developed for the study of differentiation of primary normal human bronchial epithelial (NHBE) cells by 12-0-tetradecanoyl-13-acetate (TPA) or transforming growth factor type beta (TGF-beta). Two-dimensional gel electrophoresis (2D-PAGE) of [14-C]-labeled polypeptides following treatment of NHBE cells and the human lung carcinoma cell line, A1146, with TPA and TGF-beta resulted in only quantitative polypeptide differences. NHBE cells showed a more pronounced modulation of polypeptide expression following treatment with TPA (10 nM) and TGF-beta (30 pg/ml) than A1146 cells, a cell line refractory to differentiation induction by TPA and TGF-beta. After 24 hr treatment of NHBE cells with TPA and TGF-beta, 15 polypeptides were specifically modulated by TPA, 4 were modulated by TGF-beta alone, and 6 were affected by both TPA and TGF-beta. Prolonged exposure (72 hr) resulted in predominantly (38/42) TPA-induced changes and only one polypeptide was specifically modulated by TGF-beta. Of the 42 polypeptides significantly modulated at 72 hr, 4 polypeptides (5.85/88 kDa; 6.10/40 kDa; 5.00/40 kDa; and 5.80/28 kDa) also exhibited significant quantitative modulation after 24 hr treatment.